Chronic myelogenous leukemia, sometimes referred to as chronic myeloid leukemia (CML), was the first neoplastic disease to be associated with a specific chromosomal abnormality, namely the Philadelphia or Ph.sup.1 chromosome. At the molecular level, the most notable feature is the translocation of the proto-oncogene c-abl from the long arm of chromosome 9 to the breakpoint cluster region (bcr) on chromosome 22, resulting in the formation of bcr-abl hybrid genes. The break occurs near the end of the long arm of chromosome 9 (band 9q34) and in the upper half of chromosome 22 (band 22q11).
The c-abl proto-oncogene normally encodes a protein with tyrosine kinase activity. This activity is augmented in cells carrying bcr-abl hybrid genes. The gene located at the breakpoint on chromosome 22 is called bcr because the break in chromosome 22 in CML occurs in a very small 5.8-kilobase (kb) segment (breakpoint cluster region) of the gene on chromosome 22. For purposes herein, BCR refers to the entire gene encompassing the breakpoint cluster region, while bcr shall refer to the 5.8-kb segment that is the region of the break in CML. The BCR gene is a relatively large gene of about 130 kb.
Cloning of the c-abl gene has revealed that it spans at least 230kb, and contains at least 11 exons. Two alternative first exons exist, namely exon 1a and exon 1b, which are spliced to the common splice acceptor site, exon 2. Exon 1a is 19 kb proximal to exon 2. Exon 1b, which is somewhat smaller than exon 1a, is more than 200 kb proximal to exon 2. As a result of this configuration, at least two major c-abl messages are transcribed, differing in their 5' regions. (Shtivelman et al., Cell 47, 277 (1986); Bernards et al., Mol. Cell. Biol. 7, 3231 (1987); Fainstein et al., Oncogene 4, 1477-1481 (1989)). If exon 1b is used, the mRNA is 7.0 kb. If exon 1a is used, the mRNA is 6.0 kb. Each of exons 1a and 1b are preceded by a transcriptional promotor.
The 6-kb c-abl transcript consists of exons 1a through 11. The 7-kb transcript begins with exon 1b, skips the 200 kb distance to exon 2, omits exon 1a, and joins to exons 2 through 11. Thus, both c-abl messages share a common set of 3' exons, starting from the c-abl exon 2. Consequently, the messages code for two proteins that share most of their amino acid sequence, except for the N-termini. Since the coding begins with the first exon, exonic selection will determine the protein product. The 9;22 translocation in CML results in the abnormal juxtaposition of abl sequences adjacent to bcr sequences.
The entire BCR gene has been mapped (Heisterkamp et al., Nature 315, 758 (1985)). The fusion of the BCR gene with c-abl leads to an 8.5 kb chimeric mRNA consisting of 5' BCR sequences and 3' abl sequences. The chimeric message is in turn translated into a larger chimeric abl protein (210 kDa) that has increased tyrosine kinase activity (Konopka et al., Cell 37, 1035 (1984); Kloetzer et al., Virology 140, 230 (1985); Konopka et al., Proc. Natl. Acad. Sci. U.S.A. 82, 1810 (1985)). The 210 kDa protein is considerably larger than the normal human abl protein of 145 kDa, and has a very high tyrosine kinase activity.
Two major types of bcr-abl translocations are known, characterized by two different bcr-abl junctions. One translocation is between bcr exon 2 and abl exon 2, while another translocation is between bcr exon 3 and the same abl exon 2 (Shtivelman et al., Cell 47, 277-284 (1986)). The two types of junction have been referred to as the "L-6" (or "b2a2") and "K-28" (or "b3a2") junctions, respectively. The alternative splicing from two bcr-abl exons to the abl coding sequence results in two different bcr-abl fusion proteins, one including the 25 amino acids encoded by bcr exon 3 and one which lacks those amino acids. One or both of these junctions is detected in Ph.sup.1 -positive CML patients (Shtivelman et al., Blood 69, 971 (1986)).
A significant portion of acute lymphocytic leukemia (ALL) patients carry Ph.sup.1 chromosomes in their leukemic cells. Ph.sup.1 -positive ALL is generally regarded as being less responsive to chemotherapeutic treatment than Ph.sup.1 -negative forms of ALL. This is particularly true of children with Ph.sup.1 -positive ALL.
Approximately one half of Ph.sup.1 -positive individuals afflicted with ALL express either of the two major bcr-abl junctions, L-6 or K-28. The remainder have bcr-abl genes characterized by a junction formed by the fusion of bcr exon 1 and c-abl exon 2 ("b1a2" junction). See Fainstein et al., Nature 330, 386-388 (1987).
There are thus at least three distinct bcr-abl mRNAs. The 3 mRNAs contain one of three different bcr exons fused to the same abl exon. About one half of CML patients have the b2a2 junction, while the other half are characterized by the b3a2 junction. ALL patients are about fifty percent b1a2, twenty-five percent b2a2 and twenty-five percent b3a2. An improved polymerase chain reaction (PCR) procedure has been proposed for distinguishing among the three types of molecular defects using analyses of PCR reaction products by hybridization with probes specific for the three known bcr-abl fusion sequences (Kawasaki et al., Prod. Anal. Acad. Sci. U.S.A. 85, 5698-5702 (1988)). Clinically, CML invariably progresses from the chronic phase into the blast crisis. In chronic phase CML, the increase in mature and immature myeloid elements in bone marrow and peripheral blood is the most characteristic feature (Koeffler et al., N. Engl. J. Med. 304, 201 (1981)). Kinetic studies indicate that these abnormal cells do not proliferate or mature faster than their normal counterparts. Instead, the basic defect underlying the exuberant granulopoiesis in CML appears to reside in the expansion of the myeloid progenitor cell pool in bone marrow and peripheral blood. Id. Nevertheless, the generation of terminally differentiated cells indicates that the process of hematopoiesis retains some normal features. In contrast, during blastic transformation, the leukemic cells exhibit a marked degree of differentiation arrest with a "blast" phenotype (Rosenthal et al., Am. J. Med. 63, 542 (1977)). The onset of the blastic transformation or "blast crisis" limits the therapeutic options available. The disease-free period, and consequently survival, is generally brief. Typically it is less than about four months.
The earliest treatment of CML chronic phase consisted of chemotherapy with an alkylating agent such as busulfan, and inhibitors of DNA synthesis, such as hydroxyurea. While both drugs are useful in the control of the excessive granulopoiesis of CML, their effect is not specific, since they inhibit nucleic acid synthesis in both normal and leukemic cells. With this standard approach to treatment, the median survival is about 47 months, but there is little evidence that these patients live significantly longer than patients who receive no therapy (Bergsheel, "Chronic Granulocytic Leukemia", in Fairbanks (ed.) Current Hemotology, Vol. 2, Wiley, New York, pp. 1-26 (1983)). Attempts to eliminate the leukemic clone by splenic irradiation, splenectomy and intensive chemotherapy have been observed to suppress the Ph.sup.1 -chromosome-positive population temporarily in one third of CML patients, but failed to alter the course of the disease (Cunningham et al., Blood 53, 375 (1975)). The leukemic population always recurred, resulting ultimately in blast crisis and death.
Chemotherapeutic agents such as busulfan and hydroxyurea are not specific since they inhibit nucleic acid synthesis both in normal and leukemic cells. Moreover, it is debatable whether they are .effective in altering or delaying the natural course of the disease.
More recently, interferons were added to the therapeutic armamentarium in CML chronic phase. Alpha-interferon (3-9 million units intramuscularly each day) produces a normalization of the blood count in about three quarters of chronic phase CML patients. Unlike patients treated with hydroxyurea and busulfan, more than one third of alpha-interferon treated patients have a decrease in Ph.sup.1 -chromosome-containing methaphases, and about 15% of treated patients have fewer than 5% Ph.sup.1 -positive cells. The experience with alpha-interferon is limited, since it was initially utilized in 1985. There is yet no firm evidence that the prognosis is better in interferon-treated patients than in those treated with hydroxyurea or busulfan. In addition, alpha-interferon has several side effects that include fever, anorexia, muscle and bone pain, depression, and often immune thrombocytopenia. A further disadvantage of alpha-interferon is that it is more complex to administer in comparison to hydroxyurea or busulfan (intramuscular injection versus oral intake). Although alpha-interferon preferentially affects the growth of the leukemic clone, the effect is, however, non-specific, as indicated by the persistence of Ph.sup.1 leukemic cells and the inhibition of normal hematopoietic cell growth.
In addition to chemotherapy and alpha-interferon treatment, a more rigorous therapy for CML involves marrow transplantation during chronic phase in patients who have an identical twin, a histocompatible sibling, or access to a histocompatible unrelated donor. Marrow transplantation is typically carried out following extensive chemotherapy and total body radiation to eradicate Ph.sup.1 -positive leukemia cells. There is generally a long-term survival in 45-70% of patients following marrow transplantation, however, there is a 20-40% post-transplantation mortality. Marrow transplantation is most successful if carried out early in the course of the chronic phase of the disease. About 40% of patients transplanted during the chronic phase achieve long-term survival beyond five years, free of leukemia and the Ph.sup.1 chromosome (Bergsheel, J. Cancer Res. Clin. Oncol. 116, 104-105 (1990)). Transplants during the "accelerated" or blast phases are less successful. Less than 10% of such patients survive beyond five years free of leukemia (Id.) The "accelerated" phase precedes blast transformation, and is usually characterized by a progressive loss of the capability of the leukemic clone to differentiate in mature end cells.
Autologous marrow infusion has been increasingly used in CML patients, especially those in the accelerated phase. In preparation for autologous marrow infusion, marrow cells are harvested from the affected individual, are "purged" of leukemia cells by chemical agents, and returned to the patient following extensive chemotherapy or total body radiation.
During "blast crisis", therapy is for the most part ineffective, and the disease is fatal, within at most 3-6 months. While treatments such as alpha-interferon and autologous marrow infusion are promising, they are non-specific. What is needed is a Ph.sup.1 -specific agent which selectively targets cells expressing the Philadelphia chromosome while leaving other cells intact.
Caracciolo et al., Science 245, 11007-1110 (1989) disclose inhibition of the Ph.sup.1 -positive cell line K562 utilizing the antisense oligodeoxynucleotide TACTGGCCGC TGAAGGGC (SEQ ID NO:27) which is complementary to 18 nucleotides of the second exon of c-abl. Cells of the K562 line have the b2a2 junction. While the aforesaid antisense oligomer was demonstrated effective in reducing bcr-abl protein levels, the oligomer is not specific for the bcr-abl junction, as it also hybridizes to the message from untranslocated abl.
Recently, mice infected with a defective virus carrying human bcr-abl genes have been shown to develop a CML-like syndrome (Daley et al., Science 247, 824-830 (1990); Heislerkamp et al., Nature 344, 251-253 (1990)). However, such studies utilizing artificial bcr-abl constructs to initiate a CML-like condition in transgenic animals do not indicate whether bcr-abl expression is necessary for maintenance of the established disease state, or whether inhibition of bcr-abl expression may have an impact on the disease state.